The identification of genes associated with development, differentiation, disease states, and response to cellular environment is an important step for advanced understanding of these phenomena. Specifically, effective methods for conducting genetic analysis are needed to identify and isolate those genes that are differentially expressed in various cells or under altered cell environments.
Early methods developed to identify and clone such genes were primarily based on the principle of differential or subtractive hybridization [see Proc. Natl. Acad. Sci. USA 88:2825 (1991); Nature 308:149 (1984); Proc. Natl. Acad. Sci. 84:1609 (1987); and Mol. Cell Biol. 9:1041 (1989)]. Despite the usefulness of these methods, they can only analyze a fraction of the overall changes in gene expression, require large amounts of ribonucleic acid (RNA), and are lengthy and labor intensive.
Recently, Liang and Pardee [see Science 257:967 (1992)] developed a gel-based technique that facilitates a rapid and extensive analysis of differentially-expressed messenger RNA (mRNAs). Very briefly, this technique was directed toward the identification of differentially expressed genes among the approximately 15,000 individual mRNA species in a pair of mammalian cell populations, and then recovering their complementary deoxyribonucleic acid (cDNA) and genomic clones. The general strategy was to amplify partial complementary cDNA sequences from subsets of mRNAs by reverse transcription and the polymerase chain reaction. These short sequences would then be displayed on a sequencing gel. In this technique, pairs of 10-mer primers were selected so that each would amplify cDNA from about 50 to 100 mRNAs, because this number was optimal under this technique for display on the gel [Science 257:967 (1992); and see also U.S. Pat. No. 5,262,311, the disclosure of which is incorporated in toto herein].
Several groups have successfully employed the Liang and Pardee technique to identify differentially expressed genes [see Trends Genet. 11:242 (1995); Curr. Opinion Immunol. 7:274 (1994); and Nucleic Acids Res. 19:4009 (1994)]. However, limitations associated with this technique--limitations such as the lack of quantitative correlation with mRNA abundances, a significant incidence (up to 80%) of false positive signals, variable reproducibility of the display patterns, multiple sequences migrating in the gel to produce a single signal, and under-representation and redundancy of mRNA signals [see Trends Genet. 11:242 (1995); Nucleic Acids Res. 22:5763 (1994); and FEBS Let. 351:231 (1994)]--have made it difficult to fully and correctly evaluate differential gene expression. More importantly, the size of bands is not always readily predictable from the mRNA sequence.
Amplification of cDNAs at the low primer annealing temperature of 40.degree. to 42.degree. C., a non-stringent PCR condition, as used in the Liang and Pardee technique is now considered to be a major limitation of current gel display protocols [see Trends Genet. 11:242 (1995); Biotechniques 18:842 (1995); and Biochem. Biophy. Res. Commun. 199:564 (1994)]. Adaptations of the original protocol have been reported in order to overcome some of these limitations, such as the use of one base anchored oligo-dT primer for increased representation of mRNAs [see Nucl. Acid. Res. 22:5763 (1994)], and the use of long composite primers to achieve reproducible patterns under more stringent PCR conditions [see Nucl. Acid. Res. 22:5763 (1994); and FEBS Let. 351:231 (1994)]. However, all these modifications continue to involve annealing of arbitrary primers at approximately the first few rounds of amplification 40.degree. to 42.degree. C.